甘蔗DⅢ家族蔗糖磷酸合成酶基因SofSPSDⅢ克隆及其hpRNA载体构建  

Cloning of sucrose phosphate synthase gene(SofSPSDⅢ) from sugarcane and construction of its hpRNA vector

作　　者：汪淼[1,2] 李双喜[1,2] 桂意云[1,2] 秦翠鲜[1,2] 陈忠良[1,2] 廖青[2,3] 李杨瑞[1,2] 黄东亮[1,2] 

机构地区：[1]广西农业科学院甘蔗研究所/广西甘蔗遗传改良重点实验室/农业部广西甘蔗生物技术与遗传改良重点实验室,南宁530007 [2]中国农业科学院甘蔗研究中心,南宁530007 [3]广西农业科学院农业资源与环境研究所,南宁530007

出　　处：《南方农业学报》2014年第4期532-539,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31160301);"十二五"农村领域国家科技计划项目(2013AA102604-1);广西自然科学基金项目(2011GXNSFF018002);广西农业科学院基本科研业务专项项目(桂农科2012JM06;桂农科2012YZ11;桂农科2013YM41)

摘　　要：【目的】克隆甘蔗DⅢ家族SofSPSDⅢ基因并构建其hpRNA表达载体,为研究该基因生物学功能奠定基础。【方法】通过同源克隆获得甘蔗SofSPSDⅢ基因部分序列,利用RACE技术获得其全长cDNA。扩增SofSPSDⅢ目的基因约500 bp序列,利用中间载体pHANNIBAL构建该片段的hpRNA结构,然后用Not Ⅰ将整个hpRNA结构切下,克隆到双元植物表达载体pART27上,构建该基因的hpRNA表达载体。【结果】通过同源克隆和RACE技术获得SofSPSDⅢ基因全长cDNA序列,该基因全长3252 bp,5′端UTR长度59 bp,3′端UTR长度292 bp,开放阅读框(ORF)长度2895 bp,带有真核生物典型的poly(A)尾巴(GenBank登录号:HQ117935)。该基因编码蛋白含有964个氨基酸,理论分子量108.03kDa,等电点pI=6.66;SofSPSDⅢ与SoSPS2、SbSPS和TaSPS9的氨基酸序列同源性分别为99.0%、98.9%和90.0%。构建获得SofSPSDⅢ基因的hpRNA载体pART-DⅢ-Ri。【结论】成功克隆甘蔗DⅢ家族SofSPSDⅢ基因并构建基hpRNA载体,可为下一步沉默该基因、进而解析该基因的生物学功能奠定基础。[Objective]SotSPSDIII gene from sugarcane was cloned and its hpRNA vector was constructed to provide references for studying the biological function of this gene in sugarcane. [Method]Partial fragment of SotSPSDⅢ was obtained by homologous cloning, Then the full length cDNA was obtained by rapid amplification of cDNA ends （RACE）. A fragment about 500 bp in length of SotSPSDⅢ was cloned into the ligase-based vector pHANNIBAL to generate an hpRNA structure. Then this structure was cut offwith Not I and inserted into pART27, a plant binary expression vector, to construct a hpRNA vector of SotSPSDIII. [Result]Homologous cloning and RACE technotogy were applied to isolate the full length cDNA of SotSPSDIII from sugarcane. This gene was 3252 bp in length with a 59 bp length 5＇ UTR and a 292 bp length 3＇ UTR. The OFR of this gene was 2895 bp encoding a protein of 964 amino acids. The theoretical molecular weight （MW） and isoelectric point （PI） of the protein were 108.03 kDa and 6.66, respectively. This cDNA had a typical polyA tail characterized as eukaryote （GenBank accession No. HQl17935）. The homology of amino acid sequence between SofSPSDIII and SoSPS2, SbSPS, TaSPS9 was 99.0%, 98.9% and 90.0%, respectively. The hpRNA vector of SotSPSDIII was constructed by molecular biological technique. [ Conclusion]Full length cDNA sequence of SotSPSDⅢ was cloned from sugarcane. The hpRNA vector of SotSPSDⅢ was con- structed, which laid a foundation for further study on biological function of SotSPSDIII in sugarcane.

关 键 词：甘蔗 蔗糖磷酸合成酶 SofSPSDⅢ 克隆 发夹RNA 载体构建 

分 类 号：S566.1[农业科学—作物学]